Interrupted flow rapid confirmatory immunological testing device and method

ABSTRACT

A self-contained device using a gravitationally encouraged, interrupted downward and programmed flow of fluid to provide for rapid confirmatory immunological testing (“RCIT”) in a point-of-care setting. A fluid specimen such as blood, saliva or urine is deposited into a first chamber carrying a colloidal conjugate of antigens or antibodies pathogenically specific to the condition being tested and premixed with a first measured, reactive mix buffer solution carried within an openable tank. Alternately, the buffer solution is preformulated to carry the colloidal conjugate in suspension. The premixture flows out of the first chamber toward chromatographic test strips having a single layer of uniformly dispersed porous matrix material such as polyethylene and inclined in a downward flow orientation. The flow is interrupted by a holding reservoir which is drained by siphoning, gravity and capillary forces. The delayed and regulated flow provides an incubation time for a better affinity binding of the specimen. In one embodiment, after a waiting period a bladder containing a stop-wash buffer solution is opened to flow into the reservoir and onto the strips. An absorbant pad collects excess fluid at the bottom end of the strips. It is a rapid confirmatory immunological test device having an analytical panel which can provide profile diagnostic results.

PRIOR APPLICATION

This is a continuation-in-part of U.S. patent application Ser. No.10/767,897 filed Jan. 28, 2004 now abandoned.

FIELD OF THE INVENTION

This invention relates to rapid confirmatory testing devices foranalyzing body fluids and other fluids using immunochromatography, andmore particularly to apparatuses for detection of antibodies or antigensin a point-of-care fluid test.

BACKGROUND

Over past decades, the prior art has offered several types of rapiddiagnostic testing techniques primarily for body fluids. First, were theLatex Particle Agglutination tests, then the Flow Through tests leadingto the current Lateral Flow Single Step test. However, in many settingsthese rapid tests are useful only for preliminary screening purposes,not as a confirmatory test. To this day, the Western Blot AnalyticalAssay is the only one reliably used for the confirmatory detection ofHIV infection in a clinical laboratory setting worldwide. Due to itsmulti-step manipulation and verification phases, completion of this typeof assay takes days, if not weeks. Such a delay can unfortunately leadto further propagation of infectious pathogens such as HIV or otherserious results, such as the metastasis of cancers. There is virtuallyno practical or economical confirmatory rapid diagnostic testingtechnique for use in a point-of-care setting, available in the marketplace today.

Therefore, there is a need to refine the accuracy and expedite theperformances of the common chromatographic rapid testing devices to ahigher and new level for use in early confirmatory and speedy detectionof the presence of pathogens or pathogenic conditions such as occurswith HIV infection, cancers and other disorders. Such a device could beeasily adapted to detect certain lethal viruses, bacteria or otherpathogenic antigens/antibodies in body fluids and other fluids used alsoin certain food and environmental testing, and to detect otherabnormalities in body fluids which are indicative of various cancers,cardio-vascular diseases, and/or other disorders.

SUMMARY

The instant embodiments provide a device to more rapidly conduct aconfirmatory immunoassay test for an analyte in question in apoint-of-care setting.

In one embodiment there is provided a self-contained, multi-stage,programmed, interrupted downward flow, rapid confirmatory immunologicaltesting (“RCIT”) apparatus contained in a single molded enclosure. Theimmunoassay test can be performed in either a sandwich and/orcompetitive assay format. The apparatus can carry a number ofchromatographic test strips in an inclined downward orientation formingdown-flow strips. The primary exposure of a volume-measured fluidspecimen to a specific antibody or antigen (or to a group thereof)conjugated to a label such as colloidal gold, or other type of labelsuch as colloidal carbon, latex beads, or magnetic beads, etc.(hereinafter “conjugate”) in a volume measured, reactive, bufferedsolution occurs in a first chamber before flowing on to contactdown-flow strips in a second chamber. A holding reservoir locatedbetween the first and the second chambers can temporarily restrict theflow of the mixture to allow a short period of incubation beforeproceeding with the secondary specific immunological binding reaction inthe down-flow strips. The down-flow strip is preferably not of the typeused by prior lateral flow testing methods in that it is not directlyconnected with any colloidal gold conjugate pad, but rather only linkswith the bottom of the holding reservoir at the strip's top end. Thedown-flow strips preferably include only a single layer of uniformlydispersed porous matrix material such as uniformly porous polyethylenecommercially available from Porex Corporation of Fairburn, Ga. coatedwith a number of corresponding antigenic epitopes (or polymers,proteins, polypeptides, etc.) which are immuno-determinants for ananalyte in question such as a pathogen, such as the HIV or a conditionsuch as cancer. In HIV confirmatory detection, the appearance of atleast two epitope lines on a down-flow strip will not only confirm thepresence of HIV, but will also give an analytical indication of the typeof antibodies present in the specimen which can change during differentperiods of HIV infection and Acquired Immune Deficiency Syndrome (AIDS).Additionally, the down-flow strip or strips can include a control line,working as an internal system control indicator. Therefore, any positiveresult of HIV detection shown by this RCIT method can include at leastthree (3) lines appearing in the test reaction window.

In another embodiment, a supply of aqueous buffer solution is held in asealed tank until the sample specimen has been introduced into thedevice and its cap closed. A prong in the undersurface of the cappunctures a membrane sealing the upper opening of the tank allowing thebuffer solution to be dispensed into a first chamber under atmosphericpressure. A pad at the bottom of a second chamber in contact with alower part of at least one down-flow strip absorbs the excess washbuffer that has not been retained by the strip. The strip or strips canbe positioned in an incline rather than straight vertical position inorder to reduce the height of the device. The flow out of the incubationreservoir and into the strip is prompted by a combination of siphoning,gravity and capillarity action forces.

In still another embodiment of the apparatus the enclosure has aslip-cover which is retracted to extend a spring-loaded support leg fororienting the device on an incline from horizontal. A first measuredsupply of aqueous mix buffer solution is held in a sealed tank until thesample specimen has been introduced through an inlet into the measuredfirst chamber. The solution can contain the conjugate utilizing gold orother label in suspension, or alternately lyophilized conjugate can becarried on a specialized shelf or other structure within the firstchamber. Under the push of a manipulable member, the tank seal isbroken, causing the buffer to mix with the sample, and if present thelyophilized conjugate, for enough time to form a mixture which thenflows out of the first chamber through continuous mixing into anincubation reservoir into which extends the ends of one or moredown-flow oriented test strips.

The flow out of the incubation reservoir to the down-flow strips isprompted by a combination of siphoning, gravity and capillarity actionforces. A hydrophilic absorptive pad such as a synthetic sponge isplaced at the bottom of the second chamber in contact with a lower partof the strips to absorb the fluid and encourage the siphoning action.After waiting for an adequate amount of time for the mixture to flowdown into and through the strips, a bladder containing an amount of washbuffer solution is opened under the push of another manipulable memberto wash down the remaining mixture through the strips and end thereaction at the epitope lines on the strips. The lower pad absorbs thewash buffer and, along with the angled orientation of the strips,discourages reverse flow of fluid back up into the strips. Both of themanipulable members are slidable, built-in components of the apparatus.

Apart from its unique, self-contained measured volume mix and washbuffer tanks, and its simplified program for successively opening thesetanks during processing, it is the interrupted down-flow action causedby siphoning, gravity, and to a lesser extent capillarity, from theincubation reservoir that improves accuracy and makes the apparatus wellsuited to rapid diagnostic point-of-care testing. The apparatus' abilityto detect multiple antigenic markers or specific antibodies for singleor multiple pathogens associated with one or more diseases or conditionsprovides a panel or profile based diagnosis with a high degree ofsensitivity, specificity and accuracy which closely approaches or evensurpasses the accuracy of Western Blot Analysis. Add to this itsrapidity and long term storage stability at room temperature and itbecomes an RCIT solution in the point-of-care setting.

In summary, the instant embodiments include, a flow immunoassay testingdevice for testing a fluid specimen, said device comprises a firstchamber shaped and dimensioned to accept said specimen and besubjectable to a supply of buffer solution; a second chamber holding atleast one chromatographic testing strip; and a flow-interruptingreservoir of a given capacity between said first and second chambers.The instant embodiments further include the strip or strips being heldin a non-horizontal, downward flow orientation. Some embodiments furthercomprise means for causing a flow from the reservoir onto said strip,wherein the flow is the result of a combination of siphoning,gravitational and capillarity forces acting thereon. In still otherembodiments the strip comprises a single layer of uniformly dispersedporous matrix material. In further embodiments the strip comprises anumber of quantified test lines adapted to provide a measurable basisfor a possible quantitative result display. The instant embodimentsinclude the device further comprising a colloidal conjugate specific toa condition being tested. In some embodiments an amount of quantifiedconjugate can be lyophilized and carried upon a shelf or other structurewithin the first chamber. In other embodiments the buffer solution canbe preformulated to carry an amount of said quantified conjugate insuspension. Other embodiments further comprise means for triggering thedispensing of the supply of buffer solution into the first chamber.Still other embodiments comprise a sampling well in communication withthe first chamber and having a funnel-shaped internal wall. Otherembodiments comprise an absorbing pad in contact with a bottom portionof said strip. Still other embodiments provide that the supply of buffersolution can be adjusted to create a minor overflow of said reservoir.

Other embodiments include a premix vessel containing said first chamberand a tank containing said supply of buffer solution, and an openablepassageway between said tank and said first chamber. In some embodimentsthe premix vessel can further comprise a piston having a head and beingslidingly engaged within a receptacle, wherein said head and saidreceptacle define said tank, thereby allowing a sliding translationbetween said piston and said receptacle to cause a pressurization ofsaid tank; wherein said head is interposed between said tank and saidfirst chamber. In still other embodiments the premix vessel can furthercomprise a membrane sealing said passageway; and, means for puncturingsaid membrane, thereby dispensing said amount of premix buffer solutionthrough said passageway into said first chamber. The means forpuncturing can comprise a spike extending from an inner wall of saidreceptacle, which is positioned to puncture said membrane upontranslation between said receptacle and said piston beyond a firstdistance, thereby allowing sliding movement to pressurize said tankprior to said spike puncturing said membrane. In other embodiments thepassageway can be dimensioned to cause a jet of said buffer solutioninto said first chamber.

In still other embodiments, the tank is further dimensioned to containan amount of gas in addition to said measured amount of premix buffersolution, thereby allowing volumetric compression of said tank whilesaid passageway is closed. The gas can be selected to have inert orinactive components such as nitrogen to help provide greater stabilityand a longer shelf-life at room temperature.

In still other embodiments, the apparatus further comprises an openablewash bladder shaped and dimensioned to releasably hold an amount of awash buffer, wherein an outer surface of said bladder is incommunication with said station. The wash bladder can be formed fromfrangible material. The apparatus can further comprise means forpuncturing said bladder. In some embodiments, the means for puncturingcan comprise a first spear slidingly mounted to said apparatus. Themeans for puncturing can further comprise a second spear positionedparallel to and spaced apart from said first spear. In some embodiments,the first spear has a length sufficient to puncture opposite walls ofsaid bladder. In other embodiments, the first spear has an axial airchannel.

In still other embodiments, the amount of premix buffer solution ispreferably between about 200 microliters and about 300 microliters.

Other embodiments provide an interrupted downward flow fluid specimentesting apparatus comprising: a premix vessel containing a dispensablemeasured amount of premix buffer solution and having an inlet; and atest station shaped and dimensioned to hold at least one down-flow testdevice in a non-horizontal orientation; said premix vessel furtherhaving an outlet in communication with said test station. In someembodiments, the premix vessel comprises a first chamber incommunication with said inlet; a tank shaped and dimensioned to holdsaid premix buffer solution; and an openable passageway between the tankand first chamber. The apparatus can further include a means forinterrupting flow between the vessel and station.

Still other embodiments provide a method for premixing a measured buffersolution with a fluid specimen to form a mixture for dispensing to adown-flow test device, said method including the steps of: preloadingsaid solution within a tank in a premix vessel; placing a fluid specimenin a premix chamber in said vessel; opening a passageway between saidtank and said premix chamber, thereby creating a mixture of saidsolution and said specimen; and dispensing said mixture from said vesselto said device. In some embodiments, the method can further comprisepressurizing said tank prior to opening said passageway, such as byvolumetrically compressing said tank. Alternatively, the pressurizingcan comprise forming the tank from a hollow receptacle engaged by apiston carrying the chamber; and translating the receptacle with respectto the piston. In yet other embodiments the method can further comprisewashing the down-flow test device after passage of a time period afterdispensing the mixture from the vessel.

Yet other embodiments relate to an improved immunoassay testing devicewherein a fluid specimen in a buffered solution is first contacted witha colloidal conjugate in a first part of said device, then applied to atleast one testing strip in a second part of the device; the improvementcomprising a flow interrupting reservoir between said first and saidsecond parts. The improvement can further comprise orienting the stripin a non-horizontal orientation, especially for a downward movement ofthe two interrupted but consecutive (with only a minute apart from eachother) flows. In some embodiments, the improvement further comprises anescape port in an upper part of the reservoir in communication thesecond part.

In still other embodiments, an immunoassay testing apparatus is providedwhich comprises: a first chamber; a means for exposing said firstchamber to a colloidal conjugate in a movable carriage when saidcarriage is moved a first distance; a second chamber holding at leastone down-flow testing strip; and a flow-interrupting reservoir of agiven capacity between the first and second chambers. The apparatus canfurther comprise a supply of a mix buffer solution containing theconjugate. Alternatively or additionally, the apparatus can furthercomprise a sampling well in communication with said first chamber,wherein said sampling well comprises a funnel-shaped internal wall. Insome embodiments, the means for exposing comprises: a tank holding saidsupply of buffer; a passageway between said tank and said first chamber;a membrane sealing said passageway; and means for puncturing saidmembrane, thereby dispensing said supply through said passageway. Insome embodiments, the device further comprises a vessel carrying saidtank and said carriage; and at least one spike extending from a wall ofsaid vessel toward said membrane, said spike being shaped anddimensioned to puncture said membrane when said carriage is moved saidfirst distance. Alternatively or additionally, the apparatus can furthercomprise an absorbing pad in contact with a portion of said strip. Insome embodiments, the reservoir has an escape port in an upper partthereof whereby a liquid mixture from said first chamber must accumulateinto said reservoir up to the level of said escape port before flowingfreely into said second chamber. In some embodiments, the apparatusfurther comprises filtration material in said sampling well. In someembodiments, the supply of mix buffer solution is adjusted to create aminor overflow of said reservoir. In some embodiments, the secondchamber holds a plurality of down-flow strips each having a plurality oftest lines.

Yet other embodiments relate to an improved immunoassay testing devicewherein a specimen is contacted with a movable and reactive solution,the resulting mixture is applied to at least one chromatographic teststrip, and said strip is contacted by a movable wash solution, whereinthe improvement comprises: a first built-in manipulable member forreleasing said reactive solution onto said specimen; and a second,built-in manipulable member for releasing said wash solution onto saidstrip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatical cross-sectional illustration of a firstembodiment of the interrupted flow testing device according to theinvention.

FIG. 2 is a perspective view of the device of FIG. 1.

FIG. 3 is a perspective view of a second embodiment of the interrupteddown-flow testing apparatus according to the invention in its retractedposition.

FIG. 4 is a perspective view of the interrupted down-flow testingapparatus of FIG. 3 in its extended position.

FIG. 5 is a diagrammatical cross-sectional side view of the device ofFIG. 4 taken along line 5-5.

FIG. 6 is a diagrammatical partial perspective view of the premix vesselportion of the apparatus indicating the orientation of cross-sectionplanes for FIGS. 7, 10 and 11.

FIG. 7 is a diagrammatical cross-sectional side view of a firstembodiment of the premix vessel according to the invention.

FIG. 8 is a diagrammatical partial perspective view of the wash bladderand puncturing prongs portion of the apparatus in an extended position.

FIG. 9 is a diagrammatical cross-sectional side view of the rear portionof the enclosure showing the wash buffer bladder and puncturing prongsin their pretest position.

FIG. 10 is a diagrammatical cross-sectional side view of an alternateembodiment of the premix vessel according to the invention.

FIG. 11 is a diagrammatical cross-sectional top view of anotheralternate embodiment of the premix vessel according to the invention.

DESCRIPTION

The instant device is useful to determine the presence of analyte in asample. The sample can include, for example, body fluids such as wholeblood, serum, plasma, urine, spinal fluid, amniotic fluid, mucous,saliva, and the like.

Analyte, as used herein, refers to a compound or composition to bemeasured. The analyte can be any substance for which there exists anaturally occurring specific binding member such as a binding molecule(e.g., an antibody) or for which a specific binding member such as abinding molecule can be prepared.

Analyte also includes any antigenic substances, haptens, antibodies, andcombinations thereof. The analyte can include a protein, a peptide, anamino acid, a ligand, a hormone, a steroid, a vitamin, a drug includingthose administered for therapeutic purposes as well as thoseadministered for illicit purposes, a bacterium, a virus, and metabolitesof or antibodies to any of the above substances. The analyte can alsocomprise an antigenic marker or antibody for single or multiplepathogenic conditions.

Representative analytes include steroids such as estrone, estradiol,cortisol, testosterone, progesterone, chenodeoxycholic acid, digoxin,cholic acid, digitoxin, deoxycholic acid, lithocholic acids and theester and amide derivatives thereof; vitamins such as B-12, folic acid,thyroxine, triiodothyronine, histamine, serotonin, prostaglandins suchas PGE, PGF, PGA; antiasthmatic drugs such as theophylline,antineoplastic drugs such as doxorubicin and methotrexate;antiarrhythmic drugs such as disopyramide, lidocaine, procainamide,propranolol, quinidine, N-acetylprocainamide; anticonvulsant drugs suchas phenobarbital, phenytoin, primidone, valproic acid, carbamazepine andethosuximide; antibiotics such as penicillins, cephalosporins,erythromycin, vancomycin, gentamicin, amikacin, chloramphenicol,streptomycin and tobramycin; antiarthritic drugs such as salicylate;antidepressant drugs including tricyclics such as nortriptyline,amitriptyline, imipramine and desipramine; as well as metabolitesthereof. Additional therapeutic drugs include, for example,carbamazepine, free carbamazepine, cyclosporine, digoxin, FK778,gentamicin, lithium, N-acetylprocainamide, quinidine, tacrolimus,valproic acid, free valproic acid, vancomycin, and the like, as well asthe metabolites thereof.

Representative analytes also include drugs of abuse, and theirmetabolites, including amphetamines, methamphetamines, barbiturates,benzodiazepines (BZD), cannabinoids, cocaine (benzoylecgonine), opiates,phencyclidine (PCP), tricyclic antidepressants (TCA), methadone,propoxyphene (PPX), marijuana (THC), methylenedioxymethamphetamine(MDMA, or Ecstasy, or XTC), morphine, oxycodone, and bupromorphine.Representative drugs of abuse include ethanol, heroin, hydromorphone,oxymorphone, metopon, codeine, hydrocodone, dihydrocodeine,dihydrohydroxy codeinone, pholcodine, dextromethorphan, phenazocine anddeonin.

Hepatic analytes include, for example, albumin bromocresol green (BCG)or purple (BCP), alkaline phosphatase, hepatitis B core antigen/antibody(anti-HBc), hepatitis Be antigen/antibody (anti-HBe), hepatitis Bsurface antigen/antibody (anti-HBs), hepatitis C virus (HCV), anti-HCV,direct bilirubin, gamma-glutamyl transpeptidase (GGT), antibody toHepatitis A virus (HAVAb)-IgG, HAVAb-IgM, hepatitis B surface antigen(HBsAg), lactate dehydrogenase (LD), neonatal bilirubin, prealbumin,total bilirubin, total protein, and the like.

Analytes related to pregnancy and fertility include, for example, humanchorionic gonadotropin (hCG), beta-hCG, total beta-hCG, luteinizinghormone (LH), follicle stimulating hormone (FSH), dehydroepiandrosteronesulfate (DHEAS), estradiol, free estriol, total estriol, progesterone,prolactin, sex hormone binding globulin (SHBG), testosterone, and thelike.

Analytes to determine blood disorders include, for example, B12,ferritin, folate, haptoglobin, and transferrin.

Analytes used to determine cardiac disorders include, for example,carcinoembryonic antigen (CEA), C-reactive protein (CRP), highlysensitive C-reactive protein (hsCRP), creatine kinase (CK), CK-MB,myoglobin, troponin, B-type natriuretic peptide (BNP), apolipoproteinA1, apolipoprotein B, and high density lipoprotein (HDL).

Cancer analytes include, for example, prostate specific antigen (PSA),free PSA, total PSA fecal occult blood (FOB), acid phosphatase,alphafetoprotein (AFP), beta2 microglobulin, CA 125™, CA 15-3™, CA19-9™, carcino embryonic antigen (CEA), PAP, pepsinogen, squamous cellcarcinomas (SCC), and the like.

Analytes associated with inflammation and immunology include, forexample, C3, C4, CRP, IgA, IgG, IgM, RF, and the like.

Analytes used to determine exposure to disease causative organismsinclude, for instance, rubella IgG, rubella IgM, Toxoplasmosis IgG andIgM, cytomegalovirus (CMV) IgG and IgM, HTLV III, Anti-EBNA,mononucleosis, HAA, herpes, and anti-Streptolysin O.

Infectious disease analytes include microorganisms such as Streptococcuspyogenes, Staphylococcus aureus A, Chlamydia, Syphilis, Gonococcus,Helicobacter pylori (H. pylori). Additionally, disease analytes includeviral organisms such as hepatitis (HBV, HBsAg, HCV, HAA), hepatitis Avirus, hepatitis B virus, hepatitis non Non A-Non B, IgE,cytomegalovirus (CMV), herpes viruses, rubella viruses and the like.Further included, are, for example, toxoplasmosis, anti HTLV-I/HTLV-II,BSE, chagas antibody, CMV AB, CMV IgG, CMV IgM, CSF glucose, CSFprotein, HIV AB HIV-1/HIV-2 (rDNA), rotazyme II, and the like.

Analytes pertaining to endocrinology include, for example, thyroglobulinautoantibodies (anti-Tg), thyroid peroxidase autoantibodies (anti-TPO),C-Peptide, Cortisol, HbAlc (hemoglobin fraction), PTH, Triiodothyronine(T3), free T3, total T3, thyroid hormone, Thyroxine (T4), free T4, totalT4, thyroid stimulating hormone (TSH), and the like.

Pancreatic analytes include, for example, amylase, lipase, and the like.

Veterinary analytes include, for example, Heartworm Ag, E. canis Ab,Lyme Ab, Giardia, parvovirus, FIV, FeLV, and the like.

Analytes can also include, for example, insulin, gamma globins,allergens, cystic fibrosis, toxins, such as those associated withtetanus and animal venoms, and insecticides.

The precise nature of a number of analytes together with a number ofexamples thereof are disclosed in U.S. Pat. Nos. 4,299,916, Nov. 10,1981, and 4,366,241, Dec. 28, 1982, each of which is hereby incorporatedby reference in its entirety.

The signal provided to the user of the device is provided by a bindingmember such as a specific antibody and/or antigen (“binding molecule”)conjugated to a label. Preferably labels that produce a readilydetectable signal are used. Thus, colored labels permitting visibledetection of the assay results without the addition of furthersubstances and/or without the aid of instrumentation are preferred.Examples of labels that can readily detected include, for example, dyesols, metal sols, nonmetal sols, colored latex particles, colorindicators, colored matter encapsulated in liposomes, and the like.

Metal sols are disclosed in U.S. Pat. Nos. 4,313,734, Feb. 2, 1982,4,775,636, Oct. 4, 1988, each of which is hereby incorporated byreference in its entirety, and comprise a metal, a metal compound, suchas metal oxides, metal hydroxides and metal salts, or polymer nucleicoated with a metal or metal compound. The metal sols can comprise, forexample, metals such as platinum, gold, silver and copper.Alternatively, or additionally, the metal sols can comprise metalcompounds, such as, for example, silver iodide, silver bromide, copperhydrous oxide, iron oxide, iron hydroxide or hydrous oxide, aluminumhydroxide or hydrous oxide, chromium hydroxide or hydrous oxide,vanadium oxide, arsenic sulphide, manganese hydroxide, lead sulphide,mercury sulphide, barium sulphate and titanium dioxide.

Nonmetal sols, such as carbon sols and their use are described in U.S.Pat. No. 5,559,041, Sep. 24, 1996, which is hereby incorporated byreference in its entirety. Nonmetal colloidal particles, such asselenium particles, are disclosed in U.S. Pat. No. 4,954,452, Sep. 4,1990, which is hereby incorporated by reference in its entirety. Othernonmetals that can be used include elements within Group VI A. of thePeriodic Table, sulfur, and tellurium.

Labels can also be formed from dye polymers, whereby dye molecules, orchromogenic monomers, are polymerized to form a colored polymerparticle. Examples of such dyes include Congo red, Trypan blue, andLissamine blue.

Organic polymer latex particles are disclosed in U.S. Pat. No.5,252,459, Oct. 12, 1993, which is hereby incorporated by reference inits entirety. Such particles can comprise a plurality ofnon-chromophoric monomers.

Particulate labels comprising a dye or other colored substance enclosedin liposome sacs are described in U.S. Pat. Nos. 4,703,017, Oct. 27,1987, and 5,591,645, Jan. 7, 1997, each of which is hereby incorporatedby reference in its entirety.

The devices described herein use test strips that preferably comprise adry porous material. By “porous” it is meant that the matrix is composedof a material into which fluids can flow and can easily pass through.Representative materials useful in practicing the invention describedherein, include nylon, plastic, fiber containing paper, such as filterpaper, chromatographic paper, and the like, nitrocellulose, glassfibers, polysulfone, polyvinylidene difluoride, polyurethane, and otherporous polymers, polysaccharides, (e.g., cellulose materials, such aspaper and cellulose acetate), silica, inorganic materials, such asdeactivated alumina, diatomaceous earth, MgSO₄, or other inorganicfinely divided material conveniently substantially uniformly dispersedin a porous polymer matrix, with polymers such as vinyl chloride, vinylchloride-propylene copolymer, and vinyl chloride-vinyl acetatecopolymer; cloth, both naturally occurring e.g., cotton and synthetic(e.g., nylon cloth), porous gels, (e.g., silica gel, agarose, dextran,and gelatin), polymeric films, (e.g., polyacrylamide), and the like. Inpreferred embodiments, the test strips comprise POREX CHEMISTRY A and/orPOREX CHEMISTRY K membranes commercially available from PorexCorporation, Fairburn, Ga., and/or NOVYLON brand membrane commerciallyavailable from Cuno Incorporated, Meriden, Conn.

The preferred embodiments will be described in connection with thedetection of HIV in a fluid specimen. Those skilled in the art willreadily appreciate adaptation of these embodiments to detect otheranalytes indicative of other pathogens, or pathogenic conditions withinbody, food or environmental fluid samples.

Referring now to the drawing, there is shown in FIG. 1-2 a firstembodiment of an immunoassay testing device 1 according to theinvention. The device is preferably packaged in a molded plasticenclosure 2 topped by a sealing cap 3. In the upper region of thedevice, and immediately under a ceiling hole 4 is a sampling well 5. Theinternal wall of the well is funnel-shaped, and retains some filtrationmaterial 6. The geometry of that wall, whether in the form of a V or aU, has a portion of a relatively low pitch so that when a fluid specimen7 such as whole blood, 7 or saliva runs along the wall, particles andadhesive matters are separated from the fluid component of the specimen.A supply of aqueous buffering solution 8 is held in a tank 9 along sidethe sampling well. The tank has a top opening hermetically sealed by amembrane 10, and a dispensing port 11 in a lower region leading to afirst chamber 12 in a first analytical part of the device. The chamberis located immediately below the sampling well and receives the fluidcomponent and is subjectable to the buffering solution. That firstchamber holds colloidal gold conjugated specific to the HIV antigen orother colloidal conjugate 13 specific to the condition being tested thatreacts with the fluid specimen in its buffered solution.

In some embodiments, the mix buffer comprises Borax: 2-3%; nonfat drymilk: 0.2-0.5%; Sucrose: 0.05-0.1%; NaN3: 0.4%; Rabbit IgG: 2%; GoatIgG: 2%; Human IgG: 0.2%; Tween 20: 0.5%.

An outlet 14 at the bottom of the chamber leads to an incubationreservoir 15 in which the solution flowing from the chamber accumulatesand rests until the level of the solution reaches an upper part of thereservoir where an escape port 16 leads to a second chamber 17 holdingone or more chromatographic testing strips held in a non-horizontal,downward flow orientation 18 and can be referred to as down-flow strips.

The membrane portion of the down-flow strips is in the preferredembodiments a single layer of substantially uniformly dispersed porousmatrix material such as porous polyethylene commercially available fromPorex Corporation of Fairburn, Ga. The substantially uniform dispersionof pores in the material reduces the negative effect of lot-to-lotvariation present in prior strips. This greater predictability in how agiven strip will perform enhances the ability of the device to provide aconfirmatory test. Further, such predictability allows for the creationof a quantitative assay strip having a number of lines where each lineis selected to appear at a different concentration threshold. In thisway, the device carrying one or more quantitative assay strips canprovide the digital display of a quantitative result.

Each down-flow strip 18 is preferably positioned in an inclined positionat a pitch angle A of at least 15 degrees from the horizontal. The upperedge 19 of the strip dips into the reservoir and is contacted by thesolution that flows down slowly under the effect of capillarity, gravityan siphoning forces enhanced by an absorbing pad 20 positioned in thebottom of the enclosure and in contact with the lower portion of thestrip 18. In this way the second chamber 17 and strip form a siphonbetween the reservoir 15 and the absorbing pad.

The down-flow strips are coated with a number of binding members to theanalyte. Examples include immuno-determinant epitopes of the HIV virussuch as p18, p24, p32, gp36, gp41, p51, p55, p65, gp120, gp160 andsubtype o.

Immobilization techniques include reel to reel dispenser of bindingmembers such as binding molecules. The down flow test strips are thenblocked to facilitate the test. Such a blocking procedure includes, forexample, treating the strips with a buffer comprising Triton X-100: 1%;Polyvinyl Alcohol (PVA) (30,000-70,000 mw) or Polyvinyl Pyrrolidone(PVP) (10,000 mw): 1-1.5%; and Sugar: 0.2%.

It should be noted that the escape port 16 acts as a means forrestricting the flow therethrough. Further, it should be understood thatthe dispensing of the buffer solution 8 out of the tank 9 is triggeredby puncturing the membrane 10. The puncturing is accomplished by a prong21 which extends from the underside of the cap 3 and passes through anaperture 22 in the roof of the enclosure. The prong is normally heldinto a retracted position during storage and shipment of the device, butcan be moved to an extended position by manipulating a knob 23 on theoutside of the cap. The prong is positioned, shaped and dimensioned toextend sufficiently through the aperture 22 into rupturing contact withthe membrane 10.

The buffer solution 8 washes and carries the components of the specimenthat comes down from the sampling well to form a mixture and provide thevolume of fluid necessary 15 to fill the incubation reservoir and thus,regulate the transfer of the specimen through the device. By adjustingthe volume of buffer solution to what is necessary to create a minorand/or relatively slow overflow of the reservoir, excessive flooding ofthe test strip is avoided.

It should be understood that the flow-interrupting reservoir 15 canassume a variety of positions and configurations that provides atemporary, but longer incubation time for the buffered sampling solutionto complete the first affinity binding of the immuno-chemical reactionand reach a maximum, before it is contacted with the strip to form thesecond affinity binding of the immuno-chemical reaction, forming thedouble antigen (or double-antibody in some cases) sandwich-immunocomplex. Within about 0.5-2 minutes, the relatively slow speed of thedownward flow of the mixture caused by the combination of siphoning,gravity and capillarity action forces, promotes a maximum degree of thesecond affinity binding on the down-flow strips. The two maximizedaffinity binding steps in turn maximize the diagnostic sensitivity ofthe RCIT device technology.

A transparent window 24 sealed to the enclosure provides a directviewing of chromatographic test lines 25 appearing on a number ofdown-flow test strips 26.

Referring now to FIGS. 3-5, there is shown a second embodiment of animmunoassay testing apparatus 31 according to the invention. Theapparatus is preferably packaged in a molded plastic enclosure 32 havinga base pan 33. An extendable and retractable slip-cover 34 is slidinglymounted at the back end 35 of the enclosure. The slip-cover is in aretracted position (as shown in FIG. 3) during storage and shipment ofthe device, but can be slid rearwardly to an extended position (as shownin FIG. 4) by pulling axially backward 36 on the slip-cover whileholding the base pan 33 stationary. This action also causes theextension of a spring-loaded support leg 37 hingedly mounted within arecess 38 in the bottom outer surface 39 of the enclosure. The legsupports the apparatus upon a level surface 40 so that a test station 41holding one or more down-flow testing strips 42 is oriented in aninclined position at a pitch angle A of at least 15 degrees from thehorizontal.

In the medial region of the apparatus, and immediately under a ceilinghole 45 is a sampling well 46. The internal wall 47 of the well isfunnel-shaped. The geometry of that wall, whether in the form of a V ora U, has a portion of a relatively low pitch so that when a fluidspecimen such as whole blood or saliva runs along the wall, particlesand adhesive matters are separated from the fluid component of thespecimen. Alternately, or in addition to this feature, a screen 48 canbe placed in the well to further help separate coarse particulates. Thesampling well 46 leads downwardly through an inlet 50 to a first chamber51 of a premix vessel 52 which receives the fluid component of thespecimen in a first analytical part of the apparatus.

The test is initiated by the sliding manipulation 57 of a knob 53 whichexposes and premixes the specimen with a measured amount of mix buffersolution 59 carried in an openable tank within the premix vessel. Thesolution holds in suspension an amount of colloidal gold conjugatedspecific to the HIV antigen for reaction with the specimen. In this waythe solution can be characterized as a reactive solution. The firstchamber is therefore subjectable to the mix buffer solution. In thisway, the knob and vessel form a built-in manipulable member forreleasing the mix buffer solution onto the specimen.

The amount of mix buffer solution is selected to adequately react withthe amount of sample, and in this embodiment is preferably between about200 and 300 microliters, and most preferably about 250 microliters. Themixture is dispensed through an outlet 56 at the bottom of the vesseland into a flow-interrupting incubation reservoir 65 before flowing onto one or more down-flow testing strips 42 held in the test station 41.A transparent window 60 sealed to the enclosure provides a directviewing window of down-flow test lines appearing on the strips 42.

Situated between the premix vessel 52 and the test station 41 is theincubation reservoir 65 into which the mixture briefly accumulates andrests before being drawn into a second chamber 70 housing the down-flowstrips. In the angled, test orientation, the reservoir has a givencapacity. The location and capacity of the reservoir interrupts theflow, giving the mixture more time for the first affinity binding of theimmuno-chemical reaction to occur and reach a maximum, before themixture is contacted with the down-flow strips to form the secondaffinity binding of the immuno-chemical reaction. Within about 0.5-2minutes, the relatively slow speed of the downward flow of the mixturecaused by the combination of siphoning, gravity and capillarity actionforces, promotes a maximum degree of the second affinity binding on thedown-flow strips. The two maximized affinity binding steps in turnmaximize the diagnostic sensitivity of the RCIT device technology.

The upper edge 71 of the strips dip into the reservoir 65 and arecontacted by the pooled mixture 66. The mixture flows into the strips,under the combined effect of siphoning, gravity and capillarity forces,over the upper part of the reservoir through an escape port 67 and intothe second chamber 70. Flow through the strips is enhanced by anabsorbing pad 75 positioned in the bottom front end of the enclosure andin contact with the undersurface 76 of a lower portion of the strips 42.The size of the pad is selected to accommodate the combined volume ofthe fluids within the apparatus. A block of desiccant 77 is held in athird chamber 78 in the enclosure and is in communication with thesecond chamber 70 through holes 79 to help provide a room temperaturestorage shelf life for extended periods of time, such as for at least 24months. Additionally, the entire apparatus is preferably kept in asealed plastic foil pouch bag until use.

It should be understood that the flow-interrupting reservoir 65 canassume a variety of positions and configurations that provide atemporary, but longer incubation time for the mixture to complete thefirst affinity binding.

Referring now to FIGS. 5-7, the vessel 52 is formed to have asubstantially cylindrical and axially translatable receptacle 80 engagedby substantially stationary piston 81. The head 82 of the piston and thereceptacle define an enclosed and movable tank 55 which carries ameasured supply of an aqueous mix buffer solution 59. The piston ishollow to define an internal cavity forming the first premix chamber 51.The head of the piston is interposed between and separates the tank fromthe premix chamber residing adjacent to it. A passageway 83 is formedthrough the piston head and is initially hermetically sealed by afrangible membrane 84 preferably made from a plastic laminated foil heatsealed to the outer surface of the piston head.

The fluid specimen can be, for example, body fluids such as whole blood,serum, plasma, urine, spinal fluid, amniotic fluid, mucous, saliva, andthe like. Thus the test is initiated by placing the fluid specimen,which is typically a drop of blood for HIV testing, into the premixchamber 51 through an intake aperture 85 initially positioned below thevessel inlet 50 cut through the receptacle 80 and comes to rest upon asubstantially horizontal shelf structure 88 extending from the innerwall 89 of the piston head toward a medial position in the premixchamber below the intake aperture 85 and terminating at an end 91 toform a drain 92 between the shelf end and the back wall 93 of thechamber. The shelf carries a pad 58 of porous inorganic material such asfibreglass (or 3M paper etc.) which has been impregnated with the propercolloidal gold conjugate in a lyophilized form to react with thespecimen deposited thereon. By providing the conjugate in its driedform, the device can have a self life in excess of 2 years. Theviscosity of the blood prevents it from flowing on its own through thedrain. The location and dimensions of the shelf and drain can beadjusted or further selected to adapt the apparatus to other differentviscosity fluid specimens.

The test is initiated by sliding manipulation of the knob 53 which isconnected to the receptacle 80. The sliding translation of thereceptacle over the piston 81, causes an axially channeled spike 90extending from an inner wall of the tank to puncture the membrane 84allowing the mix buffer solution 59 to be forcefully dispensed throughthe passageway 83 into the first chamber 51. Further translationvolumetrically compresses the tank 55. The dimensions of the passageway,the spike and the spike's channel can be selected to cause a jet ofbuffer fluid to be directed against the specimen causing an agitatedmixing of the buffer with the specimen to form a more thorough mixture.The lower viscosity mixture is then able to flow through the drain 92and out of the vessel. Translation moves the position of the vesselinlet 50 to close the intake aperture 85 preventing backward flow ofspecimen out of the aperture. O-rings 86,87 discourage flow of fluid inthe gap between the receptacle and piston.

It should be noted that the dispensing of the mixture out of the vessel52 is triggered by puncturing the membrane 84. A plug 96 seals a tankfill port through the end wall of the receptacle.

Referring to FIGS. 5, 8 and 9, the apparatus also optionally providesfor a supply of a stop wash buffer solution to the down-flow strips inorder to stop the reaction in the strips and to carry away lingeringchemicals and residue which could serve to obscure the lines formed onthe strips and also to remove any other non-specific materials from thereaction area. The wash buffer is preferably applied after a certainprogrammed waiting period which allows for the mixture to be drawnthrough the strips to an adequate degree. The preferred waiting time isof course dependent on the type of test being performed. For HIVdetection the waiting period is preferably between about 0.5 and 2minutes. The amount of wash buffer solution is selected to adequatelywash the down-flow strips without unduly increasing the bulk of theapparatus, and in this embodiment is preferably between about 2 and 3milliliters, and most preferably about 2.5 milliliters. In thisembodiment the volume of wash buffer is about ten times that of the mixbuffer.

For example, one such stop wash buffer comprises: Tween 20: 1%;Glycerol: 0.5%; Glycine: 5-20 mM; and NaN3: 0.02%.

The stop wash buffer solution 110 is contained in an frangible secondtank or bladder 111 located upstream from the reservoir 65. In itspretest position (as shown in FIG. 9) the bladder rests atop a pair ofprongs 120,121 extending forward from the rear wall 122 of theslip-cover 34 between a front stop 112 and a back stop 113 extendingdown from a ceiling of the enclosure. When the slip-cover 34 is moved toits extended position (as shown in FIGS. 5 and 8) the bladder falls torest on the base pan 33. The bladder is opened by re-engaging the slipcover 34 which causes the prongs 120,121 to puncture the bladder. It isunderstood that the bladder is in communication with the test station 41meaning there is a fluid path from the bladder through the reservoir tothe station. In this way, the slip-cover and prongs form a built-in,manipulable member for releasing the wash solution onto the down-flowstrips.

As shown in FIG. 8, each prong is shaped to have an axial groove 123which acts as an air channel to encourage the evacuation of the bladderwhen it is punctured. Further, the axial length L_(P) of the prongs isselected to be greater than the length L_(B) between the forward andrear axially opposite walls 124,125 of the bladder so that in there-engaged position the prongs puncture both axially opposite wallscausing the wash buffer to flow more rapidly out of the bladder whichensures rapid washing of the down-flow strips at the preselectedappropriate time.

The second down-flow of the said stop wash buffer occurs relativelyfaster than the first down-flow of the mixture. The wash buffer havingabout ten times the volume of the mixture flushes out the non-specificbinding caused by non-specific materials in the reaction area. Thisaction maximizes the specificity of the present device to provide RCIT.

Referring now to FIG. 10, there is shown an alternate embodiment of thepremix vessel 131 particularly adapted to samples having a relativelylow viscosity such as urine. This embodiment is characterized by itspremix chamber 132 having an initially sealed, openable drain 133.Further, the tank 134 formed between the receptacle 135 and the piston136 is made to be volumetrically compressible prior to the spike 137puncturing the membrane 138 sealing the passageway 139. In this way themix buffer solution 140 is pressurized prior to puncturing to causevigorous, thorough mixing. Further, the premix chamber is formed to havea limited volume which primarily acts to measure and mix the amount ofthe fluid specimen.

As the receptacle 135 is translated onto the piston 136, the receptacleacts as a movable carriage for the mix buffer tank and volumetricallycompresses the tank 134. Further translation moves the position of theinlet 142 to close the intake aperture 141 preventing backward flow ofspecimen out of the inlet. O-rings 143,144 discourage flow of fluid inthe gap between the receptacle and piston. Further translation causesthe spike 137 to puncture the membrane 138 allowing the mix buffersolution to be forcefully dispensed through the passageway 139 into thefirst chamber 132 under pressure, collapsing the tank and thoroughlymixing the buffer with the specimen to form a mixture. Once fullycollapsed, an outflow aperture 145 aligns with the vessel outlet 133 toallow evacuation the mixture therethrough. Full translation also causesthe inlet 142 to be positioned below an air vent 146 which facilitatesdownward flow of the mixture out of the premix chamber by discouragingthe formation of a vacuum.

It should be noted that the dispensing of the mixture out of the vessel131 is triggered by puncturing the membrane 138. Further, in addition tothe mix buffer supply, the tank holds an amount of a gas 147 tofacilitate the volumetric compression of the tank during translation ofthe receptacle but before the membrane is punctured. The gas can be air,or is more preferably a gas which is inert or less reactive with the mixbuffer and its suspended conjugates such as nitrogen in an effort tohelp the suspended conjugates to have a longer stability and shelf-lifeat room temperature.

Referring now to FIG. 11, there is shown a second alternate embodimentof the premix vessel 100 in which the passageway is formed by axialdepressions or channels 101,102 set into the inner surface 103 of thereceptacle 104. In this way, translation of the piston 105 beyond agiven distance will place the channels in communication with both thetank 106 and premix chamber 107 allowing the pressurized flow of the mixbuffer into the first, premix chamber to mix with the specimen depositedthrough an inlet 108 and dispensed through a drain 109.

The interrupted down-flow test can rapidly provide an analytical panelor profile of antigen or antibody detection, and confirm the biochemicalor pathogenic condition such as HIV infection, or early stage cancerprior to metastasis, or acute cardiac disorder by way of a simple,inexpensive and disposable device that can be manipulated safely by arelatively low skilled person. The above apparatus also benefits fromdry chemistry storage, for an economically prolonged room temperatureshelf life potentially in excess of 24 months.

It should be noted that the down-flow strip can be adapted especiallyfor a downward movement of two interrupted but consecutive (with onlyabout a minute apart from each other) flows utilizing a combination ofsiphoning, gravity, and to a lesser extent capillarity. Because thefirst affinity binding has already occurred in the first flow reachingthe strip, the strip does not need to be directly connected with anycolloidal conjugate pad, but only links with the incubation reservoir atits top end. Further, its membrane portion can be made from only asingle layer of uniformly dispersed porous matrix material such aspolyethylene.

The quality of the clinical performance of this novel platformtechnology surpasses any previous rapid testing technologies, such asLatex particle agglutination, Flow-Through test, and the currentlywide-spread Lateral Flow devices. It is a technology of RapidConfirmatory Immunological Testing (RCIT).

While the preferred embodiment of the invention has been described,modifications can be made and other embodiments may be devised withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

1. A flow immunoassay testing device for testing a specimen, said devicecomprises: a supply of buffer solution; an amount of conjugate specificto a condition being tested; a first chamber shaped and dimensioned toaccept said specimen and be subjectable to said supply of buffersolution to create a fluid comprising said specimen and said solution;said first chamber holding said amount of conjugate; a siphon enhancingabsorbing pad; a second chamber in fluid communication with said firstchamber, said second chamber siphoningly holding at least onechromatographic testing strip; a reservoir having a given capacity andbeing located to interrupt a flow of said fluid between said first andsecond chambers; said reservoir having an escape port in an upper partthereof; and said at least one strip having an upper edge dipping intosaid reservoir through said escape port and a downward pitch betweensaid port and a lower portion in contact with said pad to form a siphonbetween said reservoir and said pad; whereby an amount of said fluid isaccumulated in said reservoir and incubated to allow a first affinitybinding reaction to occur within said amount of said fluid to form anincubated liquid which flows slowly out of said reservoir through saidescape port and into said at least one strip under the effect ofcapillarity, gravity and siphoning forces.
 2. The device of claim 1,wherein said at least one strip comprises a single layer of uniformlydispersed porous matrix material.
 3. The device of claim 2, wherein saidat least one strip comprises a number of test lines adapted to provide ameasurable basis for quantitative result display.
 4. The device of claim1, wherein an amount of said conjugate is lyophilized and carried on astructure within said first chamber contactable by said fluid.
 5. Thedevice of claim 1, wherein said supply of buffer solution ispreformulated to carry an amount of said conjugate in suspension.
 6. Thedevice of claim 1, which further comprises means for triggering thedispensing of said supply of buffer solution into said first chamber. 7.The device of claim 1, wherein said supply of buffer solution isadjusted to create a minor overflow of said reservoir.
 8. The device ofclaim 1, which further comprises: a premix vessel containing said firstchamber and a tank containing said supply of buffer solution, and anopenable passageway between said tank and said first chamber.
 9. Thedevice of claim 8, wherein said premix vessel further comprises: saidtank comprising: a piston having a head; and said piston being slidinglyengaged within a receptacle, thereby allowing a sliding translationbetween said piston and said receptacle to cause a pressurization ofsaid tank.
 10. The device of claim 9, wherein said premix vessel furthercomprises: a membrane sealing said passageway; and, means for puncturingsaid membrane, thereby dispensing said amount of premix buffer solutionthrough said passageway into said first chamber.
 11. The device of claim10, wherein said means for puncturing comprise a spike extending from aninner wall of said receptacle, said spike being positioned to puncturesaid membrane upon translation between said receptacle and said pistonbeyond a first distance, thereby allowing sliding movement to pressurizesaid tank prior to said spike puncturing said membrane.
 12. The deviceof claim 8, wherein said passageway is dimensioned to cause a jet ofsaid supply of buffer solution into said first chamber.
 13. The deviceof claim 8, wherein said rank is further dimensioned to contain anamount of gas, thereby allowing volumetric compression of said tankwhile said passageway is closed.
 14. The device of claim 1, whichfurther comprises an openable wash bladder shaped and dimensioned toreleasably hold an amount of a wash buffer, wherein an outer surface ofsaid bladder is in communication with said second chamber.
 15. Thedevice of claim 14, wherein said wash bladder is formed from frangiblematerial.
 16. The device of claim 15, which further comprises first andsecond spears slidingly mounted to said device; said spears beingoriented and having a length sufficient to puncture opposite walls ofsaid bladder when said spears are slid into said bladder, therebyopening said bladder and releasing said amount of wash buffer.
 17. Thedevice of claim 16, wherein said first spear has an axial air channel.18. The device of claim 1, wherein said supply of buffer solution has avolume between about 200 microliters and about 300 microliters.
 19. Animmunoassay testing device comprises: a specimen; a buffer solution; aconjugate specific to a condition being tested; a first part of saiddevice for accepting said specimen, said buffer solution, and saidconjugate to create a fluid flow; said first part comprising: areservoir interrupting said fluid flow for a time sufficient to incubatea first affinity binding reaction between said conjugate and saidspecimen and thereby create an incubated liquid; a second part of saiddevice in fluid communication with said first part, wherein saidreservoir is located between said first part and said second part, saidsecond part comprising: a chamber shaped and dimensioned to siphoninglyhold at least one chromatographic testing strip in a downwardorientation, wherein an upper portion of said at least one strip dipsinto the reservoir and a lower portion of said at least one strip is incontact with a siphon enhancing absorbing pad, whereby an amount of saidincubated liquid flows into said at least one strip under the combinedeffect of capillarity, gravity and siphoning forces.